CN108445211A - 一种a族链球菌的poct荧光定量检测方法和检测试剂盒 - Google Patents
一种a族链球菌的poct荧光定量检测方法和检测试剂盒 Download PDFInfo
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Abstract
本发明公开一种A族链球菌的POCT荧光定量检测方法和检测试剂盒,所述POCT荧光定量检测试剂盒包括盒体以及设置在盒体内的上述的免疫层析试纸条,所述免疫层析试纸条包被有A族链球菌抗体,所述A族链球菌抗体为小鼠单克隆抗体或兔多克隆抗体。检测时,样本加入检测试剂盒后,经过免疫层析试纸条的层析作用,A族链球菌抗原先后与荧光微球标记抗体和检测抗体结合,最后用荧光检测仪检测荧光信号,由于荧光信号与A族链球菌抗原含量呈正相关,由此实现对咽拭子样本中A族链球菌抗原的定量检测。本检测方法的操作简单、快捷,且灵敏度和特异性高,使GAS感染者能得到及时的诊断和治疗,还能为临床治疗GAS感染者合理使用抗生素提供依据。
Description
技术领域
本申请涉及抗体领域,尤其是一种A族链球菌的POCT荧光定量检测方法和检测试剂盒。
背景技术
A族链球菌全称A族乙型(β)溶血性链球菌,英文Group A Streptococcus(简称Strep A或GAS),中文又称化脓性链球菌,是链球菌中致病力最强的细菌。A族链球菌可产生多种酶和外毒素,是引起3-15岁儿童细菌性上呼吸道感染的最常见病原体,在临床工作中极为常见。GAS感染发病后48h内如果能得到早期诊断、适当的抗生素治疗是非常有效的;若不能及时得到有效治疗,可能导致急性坏死性筋膜炎(acute necrotizing fascitis,ANF)、链球菌中毒休克综合征(streptococcal toxin shock syndrome,STSS)等严重侵袭性感染,也可引起风湿热、风湿性心脏病、急性链球菌感染后肾小球肾炎(acutepoststreptococcal glomerulonephritis,APSGN)及链球菌感染相关性儿童自身免疫性神经精神障碍(pediatric autoimmune neuropsychiatric disorders associated withstreptococcal infections,PANDAS)等并发症;而过度使用抗生素又会造成细菌耐药问题和医疗资源的浪费。
据统计,全球每年由GAS引起的急性咽峡炎为6亿1600万例。GAS相关严重疾病患者约有1810万例,且以每年178万新发病例的速度不断增加,每年至少有51.7万人死于这些严重疾病。因此,及时、正确诊断GAS急性感染极为重要。
随着人们生活水平的提高,大多数发达国家都越来越重视临床用药的依据,特别是对抗生素的合理使用。自从本世纪初开始,他们就逐步开始使用快速诊断技术对门诊病人进行病原筛查,因此,未来的GAS快速诊断产品全球市场潜力巨大。目前,国内对于GAS的鉴定主要依靠细菌培养法,需时约2~4天,无法及时诊断,且存在假阴性(即灵敏度不足)、操作复杂等缺陷。因此,建立一种简单快速灵敏特异的GAS检测方法迫在眉睫。
发明内容
本发明的目的在于,解决上述现有技术中存在的缺点和不足,提供一种A族链球菌的POCT荧光定量检测方法和检测试剂盒,以实现对A族链球菌的快速准确定量和定性检测,为GAS感染者的及时治疗、在临床治疗上合理使用抗生素等提供保障。
根据本申请的第一方面,本申请提供一种检测A族链球菌的免疫层析试纸条,其包被有A族链球菌抗体,所述A族链球菌抗体为小鼠单克隆抗体或兔多克隆抗体。
作为本申请的优选方案,所述的免疫层析试纸条包括PVC粘性底板以及固定于PVC粘性底板上的样品垫、结合垫、NC膜(硝酸纤维素膜)和吸水垫,所述结合垫贴合固定于所述样品垫的下方,所述NC膜的一端贴合固定于所述结合垫的下方、另一端与所述吸水垫相连;
所述结合垫上喷涂有荧光微球标记抗体,所述荧光微球标记抗体为采用荧光微球标记所述A族链球菌抗体的偶联物;
所述NC膜上包被有检测抗体,所述检测抗体包括所述A族链球菌抗体和羊抗鼠二抗,所述A族链球菌抗体用作检测线,所述羊抗鼠二抗用作质控线。
作为本申请的优选方案,所述结合垫上荧光微球标记抗体的喷涂量为0.05~0.5μg,所述NC膜上检测抗体的包被量为0.05~0.5μg。
作为本申请的优选方案,所述荧光微球为普通荧光微球、时间分辨荧光微球、量子点和上转发光微球中的至少一种。
作为本申请的优选方案,所述荧光微球标记抗体的制备方法,包括如下步骤:
(1)用活化液替换微球原有的保存液;
(2)加入EDC(中文名:1-(3-二甲氨基丙基)-3-乙基碳二亚胺)和Sulfo-NHS(中文名:N-羟基硫代琥珀酰亚胺)进行活化:称取一定量的EDC和Sulfo-NHS,溶解后加入微球溶液中旋转反应15~20min;
(3)去除未反应或残余的反应物;
(4)用反应液超声重悬微球,然后加入一定量的待标记A族链球菌抗体,避光反应1~4小时;
(5)去除未偶联的A族链球菌抗体;
(6)封闭微球上未偶联的反应基团:用封闭液反应0.5~1小时或用封闭液重复步骤(5);
(7)用保存液重悬微球至原体积,2~8℃保存备用。
根据本申请的第二方面,本申请提供一种检测A族链球菌的免疫层析试纸条的制备方法,包括如下步骤:
(1)用点膜仪将上述A族链球菌抗体及羊抗鼠二抗包被在NC膜上,A族链球菌抗体作为检测线,羊抗鼠二抗作为质控线,36~38℃干燥箱干燥过夜;
(2)将荧光微球标记抗体喷涂在结合垫上,常温下真空干燥2h;
(3)将NC膜、结合垫、样品垫、吸水纸及PVC粘性底板组装好后,切割成试纸条,加干燥剂于4~30℃密封保存。
优选的,所述点膜仪可以是Bio-Dot XYZ3000型点膜仪,或者其他型号的点膜仪。NC膜的干燥温度可以选用近似人体体温的37℃。结合垫的干燥温度可以选用30℃左右的常温。
根据本申请的第三方面,本申请提供一种A族链球菌的POCT荧光定量检测试剂盒,其包括盒体以及设置在盒体内的上述的免疫层析试纸条。
根据本申请的第四方面,本申请提供一种A族链球菌的POCT荧光定量检测方法,包括如下步骤:
(1)制备A族链球菌裂解液;
(2)向装有咽拭子样本的保存管中加入0.1~0.5ml的A族链球菌裂解液,涡旋震荡1~5min,室温静置5~10min,收集上清液,得到待测样品;
(3)将80~100ul的待测样品加入上述的POCT荧光定量检测试剂盒中;
(4)将加了待测样品的POCT荧光定量检测试剂盒置于荧光检测仪的孵育仓,10~15min后,荧光检测仪自动检测,读取荧光值;
(5)根据步骤(1)~(4)的方法检测至少30份经临床确认为A族链球菌均为阴性的人群的咽拭子样本,分别读取荧光值;所述A族链球菌均为阴性的人群的咽拭子样本简称A族链球菌阴性对照样品;所述A族链球菌阴性对照样品的荧光值的平均值与3倍标准方差之和即为Cut-off值;
若步骤(4)中待测样品的检测荧光值大于Cut-off值,则判断为待测样品中A族链球菌抗原为阳性;
若步骤(4)中待测样品的检测荧光值小于Cut-off值,则判断为待测样品中A族链球菌抗原为阴性。
作为本申请的优选方案,所述Cut-off值为0.17。
作为本申请的优选方案,所述A族链球菌裂解液的制备方法为:
(1)用浓度为1mM~100mM缓冲液配置体积百分比为0.1%~5%的表面活性剂;
(2)用步骤(1)配得的表面活性剂配置浓度分别为1mM~100mM醋酸溶液和10mM~1M亚硝酸钠溶液;
(3)将步骤(2)配得的醋酸溶液和亚硝酸钠溶液等比例混合均匀,即得所述A族链球菌裂解液;
所述表面活性剂为Tween 20、Triton-100和十二烷基磺酸钠中的至少一种;
所述缓冲液为硼酸盐溶液、Tris-HCL或PBS溶液。
本申请的有益效果是:样本加入样品垫后,经层析作用,A族链球菌抗原先后与荧光微球标记抗体和检测抗体结合,最后用荧光检测仪检测荧光信号,由于荧光信号与A族链球菌抗原含量呈正相关,由此实现对咽拭子样本中A族链球菌抗原的定量和定性检测。相比于现有的A族链球菌检测方法,本检测方法的操作简单、快捷,且灵敏度高、特异性高,使GAS感染者能得到及时诊断和及时有效治疗;本申请对A族链球菌的定量检测能为临床治疗GAS感染者合理使用抗生素提供依据,减少过度使用抗生素和医疗资源浪费。
附图说明
图1为本申请一种实施例的A族链球菌的POCT荧光定量检测试剂盒的结构示意图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
实施例1
本实施例1提供一种A族链球菌的POCT荧光定量检测试剂盒,如图1所示,所述POCT荧光定量检测试剂盒包括盒体1和设置在盒体1内的免疫层析试纸条。
具体的,所述免疫层析试纸条包括PVC粘性底板2以及固定于PVC粘性底板2上的样品垫3、结合垫4、NC膜5和吸水垫6,所述结合垫4贴合固定于所述样品垫3的下方,所述NC膜5的一端贴合固定于所述结合垫4的下方、另一端与所述吸水垫6相连。
具体的,所述盒体1的表面在样品垫3的上方开设有采样口7。
具体的,所述结合垫4上喷涂有荧光微球标记抗体,所述荧光微球标记抗体为采用荧光微球标记A族链球菌抗体的偶联物;所述荧光微球为普通荧光微球、时间分辨荧光微球、量子点和上转发光微球中的至少一种。
具体的,所述NC膜5上包被有检测抗体,所述检测抗体包括A族链球菌抗体和羊抗鼠二抗,所述A族链球菌抗体用作检测线8,所述羊抗鼠二抗用作质控线9。所述盒体1的表面在检测线8与质控线9上方开设有观察口10,通过该观察口10可观察到NC膜5上的反应结果。
在使用时,从采样口7将待检测样品滴加到样品垫3,在层析作用下,待检测样品依次经过样品垫3、结合垫4、NC膜5,并在NC膜5上发生免疫反应,从而可在检测线,8和质控线9上观察到相应的反应结果。
为了保证反应的充分进行,使反应结果可见,所述结合垫4上荧光微球标记抗体的喷涂量为0.05~0.5μg,所述NC膜上检测抗体的包被量为0.05~0.5μg。
本实施例中所述的A族链球菌抗体为小鼠单克隆抗体或兔多克隆抗体。
实施例2
本实施例2提供一种检测A族链球菌的免疫层析试纸条的制备方法,包括如下步骤:
(1)用Bio-Dot XYZ3000点膜仪将A族链球菌抗体及羊抗鼠二抗包被在NC膜上,A族链球菌抗体作为检测线,羊抗鼠二抗作为质控线,36-38℃干燥箱干燥过夜;所述A族链球菌抗体为小鼠单克隆抗体或兔多克隆抗体;具体的,NC膜的干燥温度可以选用近似人体体温的37℃;
(2)将荧光微球标记抗体喷涂在结合垫上,30℃真空干燥2h;
(3)将NC膜、结合垫、样品垫、吸水纸及PVC粘性底板组装好后,切割成试纸条,加干燥剂于4~30℃密封保存。
实施例3
本实施例3提供一种荧光微球标记抗体的制备方法,包括如下步骤:
(1)用活化液替换微球原有的保存液:依次进行离心、去上清液、活化液超声重悬操作;
(2)加入EDC和Sulfo-NHS进行活化:称取一定量的EDC和Sulfo-NHS,溶解后加入微球溶液中旋转反应15~20min;
(3)去除未反应或残余的反应物:依次进行离心、去上清液、超纯水超声重悬操作,重复2~3次;
(4)用反应液超声重悬微球,然后加入一定量的待标记A族链球菌抗体,避光反应1~4小时;
(5)去除未偶联的A族链球菌抗体:依次进行离心、去上清液、超声重悬操作,重复1~2次;
(6)封闭微球上未偶联的反应基团:用封闭液反应0.5~1小时或用封闭液重复步骤(5);
(7)用保存液重悬微球至原体积,2~8℃保存备用;
所述A族链球菌抗体为小鼠单克隆抗体或兔多克隆抗体。
实施例4
本实施例4提供一种A族链球菌裂解液的制备方法,具体为:
(1)用浓度为1mM~100mM的缓冲液配置体积百分比为0.1%~5%的表面活性剂;
(2)用步骤(1)配得的表面活性剂配置浓度分别为1mM~100mM的醋酸溶液和10mM~1M的亚硝酸钠溶液;
(3)将步骤(2)配得的醋酸溶液和亚硝酸钠溶液等比例混合均匀,即得所述A族链球菌裂解液;
所述表面活性剂为Tween 20、Triton-100和十二烷基磺酸钠中的至少一种;
所述缓冲液为硼酸盐溶液、Tris-HCL或PBS溶液。
实施例5
本实施例5提供一种裂解咽拭子样本的方法,具体为:
(1)用100mM的PBS溶液配置体积百分比为1%的Triton-100;
(2)用步骤(1)配得的Triton-100溶液分别配置10mM的醋酸溶液和1M的亚硝酸钠溶液;
(3)将步骤(2)配得的醋酸溶液和亚硝酸钠溶液等比例混合均匀,得到A族链球菌裂解液;
(4)将500ul的A族链球菌裂解液加入咽拭子样本保存管中,涡旋震荡2min,室温静置10min,收集上清进行检测。
实施例6
本实施例6提供一种A族链球菌的POCT荧光定量检测方法,包括如下步骤:
(1)获取新鲜的咽拭子样本;
(2)向装有咽拭子样本的保存管中加入0.1~0.5ml实施例4制备的A族链球菌裂解液,涡旋震荡1~5min,室温静置5~10min,收集上清液,得到待测样品;
(3)将80~100ul的待测样品加入实施例1的POCT荧光定量检测试剂盒中;
(4)将加了待测样品的POCT荧光定量检测试剂盒置于荧光检测仪的孵育仓,10~15min后,荧光检测仪自动检测,读取荧光值;
A族链球菌抗原会被荧光微球标记抗体标记,从而发出一定的荧光,荧光检测仪可自动检测其荧光值,由于荧光信号与A族链球菌抗原含量呈正相关,由此实现对咽拭子样本中A族链球菌抗原的定量检测。
检测结果的判读如下表:
用本发明的检测试剂盒及使用本发明的检测方法检测30份经临床确认A族链球菌为阴性的人群的咽拭子样本(以下简称正常人咽拭子样本)和15份经临床确认A族链球菌为阳性的人群的咽拭子样本(以下简称病人咽拭子样本),检测数据结果如下表1和表2所示:
表1正常人咽拭子样本的检测数据结果
1.根据表1的检测数据结果,计算出30份正常人咽拭子样本的平均值和标准方差SD,然后依据以下公式计算出Cut-off值:
Cut-off=平均值+3*标准方差SD
计算结果如下:
平均值 | 0.08 |
标准方差SD | 0.03 |
Cut-off | 0.17 |
本发明选择正常人咽拭子样本检测结果的平均值与三倍的标准方差之和作为Cut-off值,判断检测样本中A族链球菌的阴阳性。当样本的检测荧光值大于Cut-off值时,判定检测样本中A族链球菌为阳性,当样本的检测荧光值小于Cut-off值时,判定检测样本中A族链球菌为阴性。
2.根据Cut-off值判断表2病人咽拭子样本诊断结果(“+”表示检测结果阳性;“-”表示检测结果阴性)
表2病人咽拭子样本的检测数据结果
从表2的阴阳性判断结果可看出,15份病人咽拭子样本的A族链球菌检测结果均为阳性,与临床检测结果的符合率为100%,说明本发明的检测试剂盒及检测方法的灵敏度高达100%。
通过上述实验验证可知,本发明的A族链球菌检测方法的操作简单、快捷,且灵敏度高、特异性高,能及时准确地诊断出GAS感染者,使病患及时地得到治疗,降低A族链球菌感染给病患造成的危害。同时,A族链球菌抗原会被荧光微球标记抗体标记,从而发出一定的荧光,荧光检测仪可自动检测其荧光值,由于荧光信号与A族链球菌抗原含量呈正相关,由此实现对咽拭子样本中A族链球菌抗原的定量检测。本发明对A族链球菌的定量检测能为临床治疗GAS感染者合理使用抗生素提供依据,减少过度盲目使用抗生素和医疗资源浪费。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换。
Claims (10)
1.一种检测A族链球菌的免疫层析试纸条,其特征在于:包被有A族链球菌抗体,所述A族链球菌抗体为小鼠单克隆抗体或兔多克隆抗体。
2.如权利要求1所述的免疫层析试纸条,其特征在于:包括PVC粘性底板以及固定于PVC粘性底板上的样品垫、结合垫、NC膜和吸水垫,所述结合垫贴合固定于所述样品垫的下方,所述NC膜的一端贴合固定于所述结合垫的下方、另一端与所述吸水垫相连;
所述结合垫上喷涂有荧光微球标记抗体,所述荧光微球标记抗体为采用荧光微球标记权利要求1所述的A族链球菌抗体的偶联物;
所述NC膜上包被有检测抗体,所述检测抗体包括权利要求1所述的A族链球菌抗体和羊抗鼠二抗,所述A族链球菌抗体用作检测线,所述羊抗鼠二抗用作质控线。
3.如权利要求2所述的免疫层析试纸条,其特征在于:所述结合垫上荧光微球标记抗体的喷涂量为0.05~0.5μg,所述NC膜上检测抗体的包被量为0.05~0.5μg。
4.如权利要求2所述的免疫层析试纸条,其特征在于:所述荧光微球为普通荧光微球、时间分辨荧光微球、量子点和上转发光微球中的至少一种。
5.如权利要求2所述的免疫层析试纸条,其特征在于,所述荧光微球标记抗体的制备方法,包括如下步骤:
(1)用活化液替换微球原有的保存液;
(2)加入EDC和Sulfo-NHS进行活化:称取一定量的EDC和Sulfo-NHS,溶解后加入微球溶液中旋转反应15~20min;
(3)去除未反应或残余的反应物;
(4)用反应液超声重悬微球,然后加入一定量的待标记A族链球菌抗体,避光反应1~4小时;
(5)去除未偶联的A族链球菌抗体;
(6)封闭微球上未偶联的反应基团:用封闭液反应0.5~1小时或用封闭液重复步骤(5);
(7)用保存液重悬微球至原体积,2~8℃保存备用。
6.一种如权利要求1至5任一项所述的免疫层析试纸条的制备方法,其特征在于,包括如下步骤:
(1)用点膜仪将A族链球菌抗体及羊抗鼠二抗包被在NC膜上,A族链球菌抗体作为检测线,羊抗鼠二抗作为质控线,36~38℃干燥箱干燥过夜;
(2)将荧光微球标记抗体喷涂在结合垫上,常温下真空干燥2h;
(3)将NC膜、结合垫、样品垫、吸水纸及PVC粘性底板组装好后,切割成试纸条,加干燥剂于4~30℃密封保存。
7.一种A族链球菌的POCT荧光定量检测试剂盒,其特征在于:包括盒体以及设置在盒体内的如权利要求1~5任一项所述的免疫层析试纸条。
8.一种A族链球菌的POCT荧光定量检测方法,其特征在于,包括如下步骤:
(1)制备A族链球菌裂解液;
(2)向装有咽拭子样本的保存管中加入0.1~0.5ml的A族链球菌裂解液,涡旋震荡1~5min,室温静置5~10min,收集上清液,得到待测样品;
(3)将80~100ul的待测样品加入权利要求7所述的POCT荧光定量检测试剂盒;
(4)将加了待测样品的POCT荧光定量检测试剂盒置于荧光检测仪的孵育仓,10~15min后,荧光检测仪自动检测,读取荧光值;
(5)根据步骤(1)~(4)的方法检测至少30份经临床确认为A族链球菌均为阴性的人群的咽拭子样本,分别读取荧光值;所述A族链球菌均为阴性的人群的咽拭子样本简称A族链球菌阴性对照样品;所述A族链球菌阴性对照样品的荧光值的平均值与3倍标准方差之和即为Cut-off值;
若步骤(4)中待测样品的检测荧光值大于Cut-off值,则判断为待测样品中A族链球菌抗原为阳性;
若步骤(4)中待测样品的检测荧光值小于Cut-off值,则判断为待测样品中A族链球菌抗原为阴性。
9.如权利要求8所述的检测方法,其特征在于:所述Cut-off值为0.17。
10.如权利要求8所述的检测方法,其特征在于,所述A族链球菌裂解液的制备方法为:
(1)用浓度为1mM~100mM的缓冲液配置体积百分比为0.1%~5%的表面活性剂;
(2)用步骤(1)配得的表面活性剂配置浓度分别为1mM~100mM的醋酸溶液和10mM~1M的亚硝酸钠溶液;
(3)将步骤(2)配得的醋酸溶液和亚硝酸钠溶液等比例混合均匀,即得所述A族链球菌裂解液;
所述表面活性剂为Tween 20、Triton-100和十二烷基磺酸钠中的至少一种;
所述缓冲液为硼酸盐溶液、Tris-HCL或PBS溶液。
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